Breast cancer remains the most commonly diagnosed cancer among women and the second leading cause of cancer mortality in the United States (Jemal A, et al., CA Cancer J Clin; 60: 277-300 (2010)). Despite major advances in adjuvant therapy for early-stage breast cancer, patients still have a 20 to 50% chance of relapse over 10 years (Brewster A M, et al., J Natl Cancer Inst; 100: 1179-83 (2008)). Metastasis, the final step of cancer progression, is responsible for most cancer related deaths and may occur after an extraordinarily long period of time after initial diagnosis and treatment (Chiang A C, et al., N Engl J Med; 359: 2814-23 (2008), Saphner T, et al., J Clin Oncol; 14: 2738-46 (1996)). Traditionally, probability of metastases has been correlated to tumor size and nodal status in breast cancer, but recent data suggest that molecular subtype may ultimately trump all traditional prognostic factors with the basal and HER2/neu intrinsic subtypes having the worst prognosis (Perou C M, et al., Nature; 406: 747-52 (2000), Sorlie T, et al., Proc Natl Acad Sci USA; 98: 10869-74 (2001), Sorlie T, et al., Proc Natl Acad Sci USA; 100: 8418-23 (2003), Cheang M C, et al., Annu Rev Pathol; 3: 67-97 (2008)). Though great strides have been made in delineating risk factors associated with recurrence, a reliable test to herald a clinical relapse does not exist. Several serum-based tumor markers are available in clinical practice for breast cancer, however a clinical intervention when a tumor marker becomes newly abnormal is usually too late to prevent an impending relapse. Therefore, American Society of Clinical Oncology (ASCO) guidelines do not recommend routine screening with tumor markers in adjuvant breast cancer patients (Harris L, et al., J Clin Oncol; 25: 5287-312 (2007)).
It is well established that tumor angiogenesis, the process of new blood vessel formation from preexisting vasculature, as well as differentiation and migration of endothelial cells, plays a crucial role in the growth and metastasis of tumors (Folkman J., N Engl J Med; 285: 1182-6 (1971), Carmeliet P, et al., Nature; 407: 249-57 (2000)). However less is known regarding the mechanisms that allow the transition from dormant, or occult, cancer cells to overt clinical relapse in cancer patients. Emerging evidence from preclinical models suggests that tumor-derived signals stimulate the quiescent bone marrow compartment, resulting in the expansion and mobilization of bone marrow-derived (BMD) VEGFR1+ hematopoietic progenitor cells (HPCs) and VEGFR2+ endothelial progenitor cells (EPCs), among others. HPCs home to the target organ and form clusters, or pre-metastatic niches, providing a permissive local microenvironment for the recruitment of incoming tumor cells and the establishment of micrometastases. EPCs are then recruited to the periphery of the micrometastatic lesions where they modulate the angiogenic switch, the transition from avascular micrometastatic lesions to vascularized macrometastatic disease. In these murine models, inhibition of VEGFR1+ significantly reduces HPC localization to the premetastatic niche and development of metastasis. Similarly, blocking EPC mobilization strongly inhibits vasculogenesis and impairs the formation of macrometastases (Lyden D, et al., Nature Medicine; 7: 1194-201 (2001), Kaplan R N, et al., Nature; 438: 820-7 (2005), Gao D, et al., Science; 319: 195-8 (2008)).
The contribution of HPCs and EPCs to human cancer progression and pathogenesis is less well understood. HPCs have been implicated in defining the premetastatic niche in axillary lymph nodes of breast cancer patients and pelvic lymph nodes of prostate cancer patients (Kaplan R N, et al., Nature; 438: 820-7 (2005), Fujita K, et al., Cancer Sci; 100: 1047-50 (2009)). Elevated EPCs have been observed in cancer patients versus healthy controls (Taylor M, et al., Clin Cancer Res; 15: 4561-71 (2009)). EPCs have been also associated with advanced stage and worse prognosis in several hematologic and solid malignancies, and some but not all breast cancer studies (Gao D, et al., Trends Mol Med; 15: 333-43 (2009), Roodhart J M, et al., Biochim Biophys Acta; 1796: 41-9 (2009), Dome B, et al., Cancer Res; 66: 7341-7 (2006), Furstenberger G, et al., Br J Cancer; 94: 524-31 (2006), Massa M, et al., J Clin Oncol; 23: 5688-95 (2005), Richter-Ehrenstein C, et al., Breast Cancer Res Treat; 106: 343-9 (2007), Naik R P, et al., Breast Cancer Res Treat; 107: 133-8 (2008)).
It is well known that typical cancer therapeutic regimens, while effective in treating cancers, are associated with significant negative side effects. Therefore, for patients in remission or without active disease, treatment is discontinued or limited in use until progression or relapse of the cancer warrants further aggressive treatment. In patients at risk for future cancer progression or relapse, a biomarker that indicates imminent progression or relapse, while allowing sufficient time to treat the patient before progression or relapse occurs, would be highly desirable.
It is hypothesized that tumor recurrence results from residual, occult micrometastases that transition to macrometastases and become clinically detectable disease. Angiogenesis is fundamental to this process and preclinical models unequivocally demonstrate that an “angiogenic switch” must be activated to support tumor progression (Folkman J., Nat Med.; 1(1):27-31 (1995), Gao D, et al., Science.; 319(5860):195-8 (2008), Weidner N, et al., N Engl J Med.; 324:1-8 (1991), Iruela-Arispe M, et al., Thromb Hemostasis.; 78:672-7 (1997)). While there are many critical components of angiogenesis, copper is emerging as essential through experiments that demonstrate decreased endothelial cell proliferation, blood vessel formation and tumor growth with copper depletion (Badet J, et al., Proc Natl Acad Sci USA.; 86:8427-31 (1989), Brem S, et al., Am J Pathol.; 137:1121-42 (1990), Juarez J C, et al., Clin Cancer Res.; 12(16):4974-82 (2006), Hassouneh B, et al., Mol Cancer Ther.; 6(3):1039-45 (2007)). Copper appears to modulate angiogenesis through multiple mechanisms including NF-kB, HIF-1 alpha and by incorporation into copper-containing enzymes superoxide dismutase-1 (SOD1), vascular adhesion protein-1 (VAP-1) and lysyl oxidase (LOX) (11-14). Tetrathiomolybdate (TM), an oral copper chelator developed for treatment of Wilson's disease, blocks angiogenesis through inactivation of copper chaperones and decreased incorporation of copper into copper-containing enzymes (Alvarez H M, et al., Science.; 327(5963):331-4 (2010)). Copper may also play a role in migration and invasion as perinuclear copper is translocated to the leading edge of endothelial cell projections during angiogenesis (Finney L, et al., Proc Natl Acad Sci USA.; 104(7):2247-52.3 (2007)). Eventually, it is transported across the cell membrane into the extracellular space resulting in activation of proangiogenic cytokines and other molecules (Finney L, et al., Proc Natl Acad Sci USA.; 104(7):2247-52.3 (2007)). Copper chelators disrupt the organization of endothelial cells into new blood vessels by restricting the availability of extracellular copper to copper-containing enzymes critical for manufacture of a mature vascular structure (Finney L, et al., Proc Natl Acad Sci USA.; 104(7):2247-52.3 (2007)). Copper depletion in non-human primates decreases peripheral circulation of VEGFR2+ endothelial progenitor cells (EPCs), which are required for new blood vessel formation (Donate F, et al., Br J Cancer.; 98(4):776-83 (2008)). While VEGFR1+ hematopoietic progenitor cells (HPCs) and CD11b+ myeloid progenitor cells establish the premetastatic niche through remodeling of the extracellular matrix (Kaplan R N, et al., Nature.; 438(7069):820-7 (2005), Erler J T, et al., Cancer Cell.; 15(1):35-44 (2009)), colonization of the premetastatic niche by EPCs, among other cells, activates the angiogenic switch (Gao D, et al., Biochim Biophys Acta.; 1796(1):33-40 (2009)).
TM chelates copper via two distinct mechanisms. When given with food, it forms a stable complex with copper and protein and prevents absorption of copper from the gastrointestinal tract. When given between meals, it is absorbed into the blood where it binds to free copper and serum albumin. TM-bound copper is no longer available for cellular uptake and is slowly eliminated. Systemic copper depletion is measured through serum ceruloplasmin (Cp), the major extracellular copper transporter, since copper complexed with TM is detectable but not bioavailable (Gartner E M, et al., Invest New Drugs.; 27(2):159-65 (2009)). In initial phase I studies of advanced malignancy refractory to standard therapy, TM was well-tolerated and effective at inducing copper depletion with 15 of 40 patients maintaining stable disease for at least 90 days (Brewer G, Clin Ca Res.; 6:1-11 (2000)). A phase II study yielded stable disease for a median of 34.5 weeks in 13 patients with advanced renal cell carcinoma (Redman B, et al., Clin Ca Res.; 9:1666-16672 (2003)). Early-stage patients with malignant mesothelioma had a doubling of time to progression from 10 to 20 months after adjuvant TM (24). Although grade 3 or 4 hematologic toxicity occurred in up to 40% of patients, it was reversible and easily managed with dose reductions.
Several serum-based biomarkers, such as CEA and CA15-3, are currently in clinical use as indicators of cancer progression or relapse. Unfortunately, such markers herald relatively late-stage events in cancer progression, and cannot predict relapse sufficiently early in the metastatic process to enable treatment designed to halt further progression of disease. Therefore, there is an urgent need for improved methods to predict cancer progression, relapse, and response or resistance to cancer therapy, which will provide a sufficiently early signal that further treatments can be administered to prevent renewed metastasis and increased malignancy.